BIOPHYSICS

Author: GONZÁLEZ MUÑOZ DIANA M..
Year: 2003.
University: COMPLUTENSE DE MADRID [More theses of this university] [www.ucm.es].
Place of defense: FACULTAD DE FARMACIA.
Place of preparation: FACULTAD DE FARMACIA.

Summary: The development of recombinant DNA engineering, and all born techniques of molecular biology that allow you to manipulate the genome of a living organism, has enabled obtaining DNA fragments carrying the gene or genes that are desired in unlimited amounts (cloning recombinant DNA). The cloned gene expression in bacteria, Escherichia coli basically, it has been widely used in industry for the production of proteins for pharmaceutical use, and research, for research biochemical and / or structural proteins produced. The bacteria can produce a large amount of recombinant protein for quick, often at low cost, by large-scale industrial fermenters, however, in many of these processes, the product of interest deposited in the form of insoluble aggregates and inactive or in the form of inclusion body. It is therefore necessary to undertake a process of folding in vitro protein subsequent to acquire its biological activity. This thesis has dealt with the study of the replacement of urea by various additives in the in vitro folding of the BMP-2 human expressed in Escherichia coli. This protein can be characterized as a factor osteogenético that induces cell phenotypes osteoblásticos in indiferenciadas. The study of these additives was conducted by inducing a marker osteoblástico as alkaline phosphatase activity in a cell line mioblástica, specifically in the cell line C2C12. We investigated the presence of various additives evluándose concentration of the same as the concentration of protein present in the folding and compared with the results obtained in the presence of urea, which was previously established in our lab. Those conditions presented preliminary results superior to control conditions, were conducted on a larger scale in order to purify the active protein and thus establish yields. It also has made a preliminary physicochemical characterization of the rhBMP-2 in solution. It has been used as a technique to measure the intrinsic fluorescence emission of the protein, to track the distortion induced in the rhBMP-2 by different chemical agents, studies that have been completed with the characterization of the solubility of the same through spectroscopy UV-Vis.

Author: GARCÍA SÁEZ ANA JESÚS.
Year: 2004.
University: VALENCIA [More theses of this university] [www.uv.es].
Place of defense: BIBLIOTECA DEL CAMPUS DE BURJASSOT.
Place of preparation: FACULTAD DE CIENCIAS BIOLÓGICAS.

Summary: The family proteins Bcl-2 are important regulators of apotosis, and perform their functions when they are associated with cell membranes. In this paper we have addressed the characterization of the interaction of representative members of the family Bcl-xL. Bid and Bax, with lipid membranes. First identified domains of interaction with these proteins in membranes by "mapping" by elicosilación. Some of these domains are chemically synthesized, and analyzed their ability to permeabilizar membrane in model systems (vesicles unilamelares large and bicapas flat). Parallel began its structural characterization media lipid and lipidomiméticos by dicroismo move and infrared spectroscopy. The results enabled us to raise models of action.

Author: IVORRA CANO ANTONI.
Year: 2004.
University: POLITÉCNICA DE CATALUÑA [More theses of this university] [www.upc.edu].
Place of defense: aula de teleensenyament.
Place of preparation: C4 Despatx Direcció nord.

Author: GUTIÉRREZ MARTÍN M. YOLANDA.
Year: 2004.
University: EXTREMADURA [More theses of this university] [www.unex.es].
Place of defense: FACULTAD DE CIENCIAS.
Place of preparation: FACULTAD DE CIENCIAS.

Summary: In this thesis is studying the effects caused by oxidative stress induced by peroxinitrio and extracellular hydrogen peroxide on the levels of Ca2 +-free intracellular in excitable cells. First we study the modulation of pumps Ca2 +, both in the plasma membrane vesicles of sinaptosomas (PMCA) and vesicles of sarcoplásmico reticulum of skeletal muscle (SERCA) induced oxidative stress generated by exposure to ONOO-. To study the homeostasis of Ca2 + cytosolic in excitable cells exposed to SIN-1, as an agent releasing ONOO- were used as a model system: granular cells of the cerebellum (CGC) and myo-tubes differentiated from the myoblast cell line C2C12 . From the results we can say that: * The treatment of plasma membrane vesicles of sinaptosomas with ONOO- inhibits the activity of the PMCA. This also inhibits oxidative activity Ca2 +-ATPase of reticulum sarcoplasmático. Inhibition of these activities involves a loss of the ability to transport ions Ca2 +, which can contribute to the deregulation of the Ca2 + intracellular homeostasis. * An exhibit of these vesicles to ONOO- results in oxidation of thiol groups and nitración waste tirosinas. Using the Ca2 +-ATPase of reticulum sarcoplasmático we found that the main target of ONOO- is located at its cytosolic domain. Physiological concentrations of reducing agents such as NADH, reduced ascorbate and glutathione protect the Ca2 +-ATPase of reticulum sarcoplasmático of ONOO-. * Channels of Ca2 +-activated L-type voltage granular cells of the cerebellum sonaltamente sensitive to oxidative species / nitrosativas generated during decomposition and SIN-1. The Ca2 +-ATPase of plasma membrane was inhibited at concentrations above these reactive species, thus altering the homeostasis of Ca2 + depend on the intensity of oxidative stress. * Myo-tubes C2C12 have channels of Ca2 +-activated intracellular deposits emptying submitting pharmacological properties different from those of the dihydropyridine-sensitive channels. These properties are identical to those described for the channels SOCs (Store-operated calcium channels). Oxidative stress / nitrosativo extracellular generated during the decomposition of INS-1 relentiza entry capacitativa Ca2 +-mediated channels SOCs.

Author: PÉREZ LÓPEZ SILVIA.
Year: 2005.
University: BARCELONA [More theses of this university] [www.ub.es].
Place of defense: FACULTAT DE FARMACIA.
Place of preparation: UNIVERSIDAD DE BARCELONA.

Summary: This thesis is structured into two distinct parts. The first part has been studied a synthetic peptide, multimérica structure, the MAP [VP1 + VP3], which contains potentially antigenic sites of viral proteins VP1 and VP3 Hepatitis A and the second, synthetic peptides corresponding to the protein E1 virus GBV-C/HGV or Hepatitis G virus, and their interaction with membrane models. The main objectives of this first part of the doctoral thesis have been studying the ability inmunógena peptide MAP [VP1 + VP3] with two different formulations that were administered to experimental animals, and valuing the differences that have been achieved in terms of terms greed and recognition epítopos constituent of the macromolecule using two different techniques, an immunoassay technique is the classical as ELISA, and another novel is based on the resonance of plasmón surface (RPS). During 98 days were followed by an immunization protocol, in which rabbits 1 and 2 were immunized with a peptide MAP [VP1 + VP3], Al rabbit 1 was administered with a formulation of Freund's adjuvant, and the rabbit 2 a formulation with liposomes MLV. In the rabbit 1 the total antibody levels remained high until the last immunization, which noted a decline, perhaps due to an effect of tolerance. In the rabbit 2 levels of antibodies were growing up to a peak of 98 days. In both cases were measured levels of antibodies to the month of the last immunization and noted that in the case of rabbit 1 had fallen virtually to zero, and not in the case of rabbit 2. In analyzing the greed were used two different techniques, the ELISA and technology RPS. In our studies, the experimental curves made to calculate the greed from ELISA were not adjusted completely to the theoretical curves of the model followed, designed to monoclonal antibodies. This discrepancy between the theoretical model and the pilot was attributable both to the complexity of antigen MAP [VP1 + VP3], as the analyte (antibody). The antigen is a peptide complex, with at least 3 sites reagents, VP1, VP3 and conformation MAP [VP1 + VP3] and the antibody can react with mono or bivalencia. In trials of RPS, one of the most troubling was the finding a mathematical approach that could adapt to our model. The MAP [VP1 + VP3] containing more than one epítopo, as seen in the ELISA, which is already recognized both the construction MAP [VP1 + VP3] as a linear peptides. In addition, the IgG could interact with the ligand with 1 or 2 domains F ab each of the epítopos, making the model more complex still. This was probably the reason why none of the analytical mathematical models to adapt fully to our data. However, considering the dissociation constants, which are inversely proportional to the greed and independent of the concentration under conditions of saturation, comparisons could be made. Both the analysis with ELISA with RPS indicated that the liposomes antiserum produced a more avidly than the Freund adjuvant. In the second part of the thesis, has been synthesized and studied synthetic peptides corresponding to the protein envelope E1 Hepatitis G (GBV-C/HGV). The GBV-C/HGV, recently discovered, has been identified as a potential inhibitor replication of the AIDS virus (HIV). The use of model membrane and the ability of peptides synthesized interact with them using different techniques, has been the main objective of this second part. As protein was selected to study the E1 virus GBV-C/HGV, which belongs to the structural proteins of the virus. There are no known studies to date today on this protein, up 8, to ebc l be structural protein should have significant sites in its antigenicity stream. In synthesize peptides, we selected different strains of GBV-C/HGV and compared them with the help of the program Clustalw to get the conserved sequence of which depart for the synthesis. The conclusion reached was that the sequence showed the best features in terms antigenicity, turns the beta, and hidrofilia was comprised between amino acids 53-66. Another method was also used to analyze the regions hidrobóbicas of protein E1. The method selected was an algorithm called Predict Protein on the basis Swiss Protein. The profile of the 190 aa protein E1 was analyzed by the program PHD (profile fed neural network systems from HeiDelberg). Around the region corresponding to the amino acids 53-66 selected, be found, according to the algorithm prediction PHDhtm, a transmembrane helix. The algorithm PiMohtm also confirmed the presence of the propeller. Because it was thought derivatizar the peptide E1 (53-66) with a fatty acid, palmitic acid, because in this way it could attempt to mimic the native hydrophobic environment in which he was. Because the protein extremes are described as potentially inmunogénicos, since the protein E1 virus has been little estudiad, it was decided that sinetizaría also a peptide corresponding to the amino-terminal end. To that end, and then to follow the same process that was followed for selection of peptide E1 (53-66), we selected amino acids between positions 3 and 17, to obtain a peptide of 15 aa. It valued the interaction of peptides synthesized model membrane through various techniques: the Langmuir isotherm, fluorescence, differential scanning calorimetry and Brewster angle microscopy. The peptides E1 (53-66), E1 (3-14) Gy E1 (3-17) R incorporates weakly in topcoat in a different electrical charge. However, the penetration was more prominent in anionic topcoat, which implies the existence of an electrostatic component that can be explained by the nature of cationic peptides to both pH experiments (7.4). The PalmE1 (53-66) joins topcoat in a different electric charge: zwitteriónica (DPPC and DOPC) and anion (DPPG and DOPG). The results obtained by the test of union isotherms indicated that the interaction presents the sequence E1 (53-66) with lipids studied, it is basically due to the presence of positive charges peptide. For its derivative palmitoílo, interaction was greater with DOPC lipids and DPCC (zwitteriónicos) with anionic lipids. The results obtained by differential scanning calorimetry indicated that the sequence E1 (53-66) had a higher ability to interact with the liposomoas MLVs of DPPC DPPG and that the sequences belonging to the party amino-terminal of protein evoltura E1. And the results of the test of union isotherms indicated that the interaction presents the sequence E1 (53-66) with lipids studied, it is basically due to the presence of positive charges peptide. For its derivative palmitoílo, the interaction is more with DOPC lipids and DPPC (Zwitteriónicos) with anionic lipids.

Author: OLASAGASTI ARSUAGA FÉLIX.
Year: 2005.
University: COMPLUTENSE DE MADRID [More theses of this university] [www.ucm.es].
Place of defense: FACULTAD DE CC. QUÍMICAS.
Place of preparation: FACULTAD DE CC. QUÍMICAS.

Summary: The thesis presents and discusses a theoretical model of the cellular system automantenido that is consistent with the laws established by the thermodynamic energy. The model presents a system of reactions that form the building blocks of membranes, as well as elements necessary for their self energy. The study was performed using a formalism of differential equations that is established as a result of a thorough analysis of the network estequiométrico reactions. The thesis presents the results of research conducted with two experimental models that support the assumptions made in the theoretical model. On the one hand, we studied the transport of protons in vesicles giant vesicles because they are perfect for the study of systems reactions encapsulated and protons can be one of the elements responsible for cell stability in the theoretical model. On the other hand, explores the possible abiotic synthesis of acidic ribonucleicos as elements capable of carrying out catalytic process in the cellular system. In this way, combining the theoretical and experimental methodology, which addresses issues stadiums canned organization of the field, prior to the formation of the earliest living things, and responds to key issues in the latter stages of the chemical evolution .

Author: MONTES BURGOS LIDIA RUTH.
Year: 2005.
University: PAÍS VASCO [More theses of this university] [www.ehu.es].
Place of defense: FACULTAD DE CIENCIA Y TECNOLOGÍA.
Place of preparation: DEPARTAMENTO DE BIOQUÍMICA Y BIOLOGÍA MOLECULAR.

Summary: This thesis has as its primary objective the effects of fosoflipasas C esfingomielinasas on the structural and functional properties of bicapas lipid. This will have used various enzymes of microbial origin, which hydrolyzed esfingomielina and, in some cases, also glicerofosfolípidos, generating respectively ceramides and diacilgliceroles. It has studied the content and release permeabilización vesicular induced ceramides, or generated in situ (by the enzymatic hydrolysis dela esfingomielina), or added to the membranes. This has been used as LUV system with different compositions of phospholipids and cholesterol, containing fluorescent molecules trapped inside it. In most experiments have used small quantities of ceramides, which may exist under physiological conditions dependent signaling ceramides. Two properties of ceramides are important to the process of restructuring the membranes; its ability to induce membrane negative bends and their tendency to segregate laterally in the plane of the membrane, resulting in rich domains ceramides. Listeria monocytogenes is a bacteria that causes infections in humans and animals. The bacterium secretes a PLC active on several types of glicerofosfolípidos (PC and SM), which can be defined more appropriately as a esfingomielinasa / phospholipase C. The enzyme activity is accompanied by major changes in the structure of the vesicles, such as: aggregation and mix and lipid content intervesiculares and is not releasing content. These results suggest that PLC LM induce fusion of vesicles. PlcHR2 is an enzyme that is part of a new superfamily fosfolipasas C / phosphatases, with members in a variety of bacterial species. We have described the hydrolytic activity of PlcHR2 when the substrate is in the form of LUV. PlcHR2 hydrolyzed PC and SM, but was inactive on other lipids. Calcium does not alter its hydrolytic activity. The enzyme inducing fusion of vesicles, and the Ca2 + does not affect the merger, but adds to the speed of aggregation and the speed and breadth of the release of contents. On the other hand, we have conducted a series of experiments aimed at understanding the hemolytic activity of PlcHR2, and try to explain its molecular mechanism.

Author: CUESTA MATARREDONA EDUARD.
Year: 2005.
University: BARCELONA [More theses of this university] [www.ub.es].
Place of defense: UNIVERSIDAD DE BARCELONA, IDIBELL.
Place of preparation: UNIVERSITAT DE BARCELONA, IDIBELL.

Summary: OBJECTIVE Fructose 1,6-bisfosfat (F1 6BP) protects the body against many processes that cause inflammation. Hipotetizamos that the primary effect of the F1 6BP is to prevent the activation of macrophages and secretion of cytokines. Our intention was to identify targets and cellular holders of this sugar bifosforilado and investigate in detail its mechanism of action. INTERVENTIONS The protective action of F1 6BP was tested in rats Saprague-Dawley (between 200 and 250 grams in weight) to which they had been induced hepatitis through the invention of galacctosamina (GalN). The in vivo effects of F1 6BP were evaluated by determining the plasma transaminase activity, the determination of endotoxins in plasma and the determination of tumor necrosis factor-alpha (TNF-alpha) in plasma and liver in rats treated Total with GalN. The targets of the F1 6BP to reduce the response of macrophages to lipopolisacrádio LPS were determined by the study of the correlation between changes in the production of TNF-alpha and the flow of potassium through the cell membrane in primary cultures of Kupffer cells. DETERMINATIONS AND MAIN RESULTS resutlados obtained in vivo experiments indicate that treatment with F1 6BP prevents damage caused by the administration deGalN in cells parènquima liver. This protection has been associated primarily to the reduction in the inflammatory response. FF1, 6BP prevent endotoxemia induced GalN rat and this correlates with the inhibition of secretion of histamine from peritoneal mast cells, an event that precedes the increase of endotoxins in plasma and liver in the production of TNF-alpha. Moreover, treatment with F1 6BP reduces the sensitivity of macrophages to LPS, which causes a decrease in the production of TNF-alpha. In vitro results show that F1 6BP inhibits the response and secretion of TNF-alpha by the Kupffer cells to be administered LPS. The F1 6BP prevent these effects by inhibiting the activation of potassium channels by LPS in this cell model. CONCLUSIONS The modulatory effect of potassium channels in the exogenous addition of F1 6BP explains its effects as antihistamine and anti-inflammatory, which greatly increases the therapeutic interest of this compound bifosforilado.

Author: DOMENECH CABRERA OSCAR.
Year: 2006.
University: BARCELONA [More theses of this university] [www.ub.es].
Place of defense: FACULTAD DE FÍSICA Y QUÍMICA.
Place of preparation: FACULTAD DE QUÍMICA.

Summary: The main purpose of this thesis has been the study of the physicochemical properties of the inner membrane of mitochondria and the interaction with cytochrome c model membrane. To achieve various techniques were used: topcoat of Langmur and Langmuir-Blodgett films, fluorescence spectroscopy, microscopy of Brewster Angle and Atomic Force Microscopy. The overall conclusion of this thesis has been: "In resolving the calcium induces phases H II in the sample POPE: CL (0.8: 0.2, mol: mol). Composition This is the single most stable both fostolípidos and corresponds to the molar fraction in the inner membrane of mitochondria. Extending these reverse micelles formed bicapas flat on a solid support, showing segregation lateral domains enriched in LC in which cytochrome c joined specifically. In this model the inner membrane of mitochondria the POPC would behave as a spacer in the domains of enriched POPE forming the lipid matrix. " The reverse process would be an explanation for the release of cytochrome c from the inner membrane of mitochondria during apoptosis.